Enzymes involved in sulfur-oxidation and transfer are increasingly being recognized as potential drug targets for development of antimicrobials, therapies for cancer, and inflammatory disease. Thiol dioxygenases catalyze the O2-dependent oxidation of thiol-bearing amino acid derivatives using a mononuclear non-heme iron site. This AREA proposal represents a continuation of our ongoing efforts to develop a `molecular level' understanding for an emerging subset of non-heme mononuclear iron enzymes involved in sulfur-oxidation. Comparative studies on two thiol dioxygenases, Mus musculus cysteine dioxygenase (Mm CDO) and a promiscuous 3-mercaptopropionic acid dioxygenase isolated from Azotobacter vinelandii (Av MDO), reveal that the denticity of substrate Fe-coordination is divergent. This perturbation to the Fe-coordination sphere significantly alters the Fe-site electronic structure (spin-state) and reactivity upon treatment with nitric oxide (NO). LC-MS/MS analysis of NO-treated samples of cysteine-bound Mm CDO (termed Mm ES-NO) identify fragmentation ions consistent with formation of a sulfur-nitrogen bond; suggesting the formation of cysteine sulfinamide. To our knowledge this `dioxygenase'-like NO-reactivity has not been previously observed for any other non-heme iron enzyme. Conversely, this product is not present in equivalent samples of Av ES-NO. We speculate that the absence of this activity for Av ES-NO is related to the differential spin-state relative to Mm ES-NO. We further demonstrate that changes to outer Fe-coordination sphere alter substrate-coordination for Av MDO to favor bidentate cys-coordination and formation of an Av ES-NO spin-state equivalent to Mm ES-NO. This provides a framework to interrogate role of the iron-nitrosyl spin-state on the observed `dioxygenase'-like NO-reactivity within a single enzyme. Specific Aim 1 presents kinetic, structural, and spectroscopic experiments designed to explore this unprecedented NO-reactivity. These studies allow for the direct interrogation substrate-bound iron-nitrosyl reactivity and may provide insight into reactions with molecular oxygen. Specific Aim 2 outlines a strategy for the development of a thiol dioxygenase kinetic mechanism by pre-steady state kinetic methods. Preliminary single-turnover and stopped-flow results are provided to demonstrate feasibility and evaluate an optimal time-window for trapping transient Fe-oxo intermediates by rapid-mix freeze-quench (RFQ) techniques. Support for this work at UTA is enthusiastic as it falls within the scope of `health and the human condition', one of the four main themes in the UTA Strategic plan. The scope of work proposed provides ample multidisciplinary training opportunities for students and includes cross-training with the Hendrich group (Carnegie Mellon University), a stated collaborator on this grant.